JP7404432B2 - Metal oxide encapsulated drug composition and method for its preparation - Google Patents

Description

This disclosure relates to pharmaceutical compositions and methods for preparing metal oxide encapsulated drugs at process temperatures of 35°C or less.

Drug-containing medicaments that have improved flowability, extended shelf life, improved solubility, and a high fraction of the drug that is functional before or after administration of the pharmaceutical composition to a subject in need thereof. Compositions (e.g., small molecules, viral particles, polypeptides, polynucleotides, mixtures of polypeptides and lipids, or mixtures of polynucleotides and lipids, small molecules, viral particles, polypeptides, polynucleotides, mixtures of polypeptides and lipids) , or mixtures of polynucleotides and lipids) is important for the pharmaceutical industry. These properties have the potential to reduce associated manufacturing costs per therapeutic dose. These properties may also (i) provide or enhance the safety, predictability, and success rate of the preparation process; (ii) improve performance over time, e.g., during preparation of the pharmaceutical composition and/or in storage conditions prior to administration (iii) increasing the solubility of the drug; and/or (iv) pharmaceutical compositions that must be administered to subjects in need of providing one or more therapeutic benefits. may result in increased commercial value of the pharmaceutical composition or increased likelihood of government approval. A number of coating techniques have been developed for encapsulating drugs, such as polymer mesh coatings, pan coatings, aerosol coatings, fluidized bed reactor coatings, molecular layer deposition coatings, and atomic layer deposition coatings. Despite advances in compositions and methods for preparing encapsulated drugs, pharmaceutical compositions prepared by known methods exhibit reduced flowability and/or degrade during the preparation process, for example. Therefore, there is an unmet need for new compositions and methods for preparing encapsulated drugs. The present invention specifically addresses this need for metal oxide encapsulated drugs.

In one aspect, a method of preparing a pharmaceutical composition having a drug-containing core surrounded by one or more metal oxide materials is provided. The method includes: (a) loading particles containing a drug into a reactor; (b) applying a vapor or gaseous metal precursor to the particles within the reactor; and (c) using an inert gas to (d) applying a vapor or gaseous oxidizer to the particles in the reactor; and (e) using an inert gas to purge one or more of the reactors. and performing a pump purge cycle. The temperature of the particles does not exceed 35°C. This produces a pharmaceutical composition that includes a drug-containing core surrounded by one or more metal oxide materials.

Implementations may include one or more of the following features.

The temperature inside the reactor need not exceed 35°C.

The series of steps (b)-(e) can be repeated one or more times to increase the overall thickness of the one or more metal oxide materials surrounding the core.

The reactor pressure may stabilize the following steps (a), (b), and/or (d).

The contents of the reactor may be agitated before and/or during step (b), step (c), and/or step (e).

A subset of the vapor or gaseous contents may be pumped out before step (c) and/or step (e).

The metal oxide layer can have a thickness ranging from 0.1 nm to 100 nm.

The particles can include a drug and one or more pharmaceutically acceptable excipients.

The particles may have a median particle size between 0.1 μm and 1000 μm, based on a volume average.

The pharmaceutical composition can be removed from the reactor and mixed with a pharmaceutically acceptable diluent or carrier.

The particles may consist essentially of drug.

The drug can be a small molecule, a viral particle, a polypeptide, a polynucleotide, a composition comprising a polypeptide and a lipid, or a composition comprising a polynucleotide and a lipid.

The one or more metal oxide materials may include aluminum oxide, titanium oxide, iron oxide, gallium oxide, magnesium oxide, zinc oxide, niobium oxide, hafnium oxide, tantalum oxide, lanthanum oxide, and/or zirconium dioxide. .

The one or more metal oxide materials may be comprised of aluminum oxide and/or titanium oxide.

The oxidizing agent can be selected from the group of water, ozone, and organic peroxides.

A polypeptide can be an antibody or an antibody fragment.

The antibody or antibody fragment may be selected from the group of alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamicin, ipilimumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, or trastuzumab.

The small molecule drug may be selected from the group of acetaminophen, clarithromycin, azithromycin, ibuprofen, fluticasone propionate, salmeterol, pazopanib HCl, palbociclib, or amoxicillin-clavulanate potassium.

In another aspect, a pharmaceutical composition having a drug-containing core surrounded by one or more metal oxide materials can be prepared by any of the methods described above.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Schematic diagram of a rotating reactor for ALD and/or CVD coating of particles, e.g. drugs Table showing typical process conditions for this method Graph showing representative residual gas analysis traces measured during steps (d), (h), (i), and (m) for one cycle of the method. (Top): Shows overlaid HPLC chromatograms of clarithromycin from compositions containing uncoated clarithromycin, aluminum oxide coated clarithromycin, or titanium oxide coated clarithromycin. Graph; (bottom): Table showing quantified values from HPLC chromatogram (Top): Overlaid HPLC chromatograms of clarithromycin carbomer complex (CCC) from compositions containing uncoated CCC, aluminum oxide coated CCC, or titanium oxide coated CCC. Graph; (bottom): Table showing quantified values from HPLC chromatogram (Top): Graph of overlaid HPLC chromatograms of pazopanib from compositions containing uncoated pazopanib, pazopanib coated with aluminum oxide, or pazopanib coated with titanium oxide analyzed; (bottom): Table showing quantified values from HPLC chromatograms (Top): Graph of overlaid HPLC chromatograms of palbociclib from compositions containing uncoated clarithromycin palbociclib, palbociclib coated with aluminum oxide, or palbociclib coated with titanium oxide; (bottom) ): Table showing quantified values from HPLC chromatogram Graph showing MALDI-TOF analyzed spectral patterns of pazopanib HCl from compositions of uncoated pazopanib HCl, pazopanib HCl coated with aluminum oxide, or pazopanib HCl coated with titanium oxide. Graph showing fragmentation patterns analyzed by MALDI MS/MS of pazopanib HCl from compositions of uncoated pazopanib HCl, pazopanib HCl coated with aluminum oxide, or pazopanib HCl coated with titanium oxide. Graph showing MALDI-TOF analyzed spectral patterns of palbociclib from compositions of uncoated palbociclib, palbociclib coated with aluminum oxide, or palbociclib coated with titanium oxide. Graph showing fragmentation patterns analyzed by MALDI MS/MS of palbociclib from compositions of uncoated palbociclib, palbociclib coated with aluminum oxide, or palbociclib coated with titanium oxide. Graph showing the relative release rate of clarithromycin over time from uncoated clarithromycin, aluminum oxide coated clarithromycin, or titanium oxide coated clarithromycin as analyzed by UV spectroscopy. Figure 2 shows the relative release rate of clarithromycin carbomer complex (CCC) over time from uncoated CCC, aluminum oxide coated CCC, or titanium oxide coated CCC as analyzed by UV spectroscopy. graph Graph showing the relative release rate of palbociclib over time from uncoated palbociclib, aluminum oxide coated palbociclib, or titanium oxide coated palbociclib as analyzed by UV spectroscopy. Graph showing the crystallinity of compositions containing uncoated indomethacin, aluminum oxide coated indomethacin, or titanium oxide coated indomethacin with and without exposure to 90% relative humidity. Representative images obtained by transmission electron microscopy of acetaminophen coated with metal oxide materials Graph showing strength versus binding energy profiles for uncoated acetaminophen or acetaminophen compositions coated with metal oxide materials as determined by XPS analysis; insets are C1s, O1s quantified from the graph. , and the percentage of N1s. Graph showing extracted ion chromatograms of intact mass of Avastin® from compositions of uncoated Avastin® or titanium oxide coated Avastin® Graph showing deconvoluted masses of predicted glycoforms and major heterogeneities detected in uncoated Avastin® or titanium oxide coated Avastin® compositions. Table showing the main heterogeneities detected by LC-MS of uncoated Avastin® or titanium oxide coated Avastin® compositions. Graph showing extracted ion chromatograms of intact mass of Herceptin® from compositions of uncoated Herceptin® or aluminum oxide coated Herceptin® Graph showing deconvoluted masses of predicted glycoforms and major heterogeneities detected in compositions of uncoated Herceptin® or aluminum oxide coated Herceptin® Table showing the main heterogeneities detected by LC-MS of uncoated Herceptin® or aluminum oxide coated Herceptin® compositions. Percentage of sequence coverage of double digest compositions of uncoated Avastin® or titanium oxide coated Avastin® compared to in-silico digested Avastin® sequences. Table showing Plot showing total compounds identified in compositions of uncoated Avastin® or titanium oxide coated Avastin® Sequence coverage of double digest compositions of uncoated Herceptin® or aluminum oxide coated Herceptin® compared to in-silico digested Aherceptin® sequences. Table showing percentages Plot showing total compounds identified in compositions of uncoated Herceptin® or aluminum oxide coated Herceptin® Graph showing the results of FTIR analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions Graph showing the results of FTIR analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions Table showing the percentages of Avastin® alpha helices, beta sheets, random coils, and beta turns from compositions of uncoated Avastin® or titanium oxide coated Avastin® Graph showing the results of far-UV CD analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions Graph showing the results of intrinsic and extrinsic fluorescence analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions Table showing λmax of uncoated Avastin® or titanium oxide coated Avastin® compositions Graph showing the results of FTIR analysis of uncoated Herceptin® or aluminum oxide coated Herceptin® compositions Graph showing the results of FTIR analysis of uncoated Herceptin® or aluminum oxide coated Herceptin® compositions Table showing the percentage of alpha helices, beta sheets, random coils, and beta turns of Herceptin® from uncoated Herceptin® or aluminum oxide coated Herceptin® compositions. Graph showing the results of deep UV CD analysis of uncoated Herceptin® or aluminum oxide coated Herceptin® compositions Graph showing the results of near-UV CD analysis of compositions of uncoated Herceptin® or Herceptin® coated with aluminum oxide (Top): Graph showing the results of size exclusion chromatography analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions; (Bottom): Quantified monomer and Table showing retention times of aggregates Graph showing the results of ion exchange chromatography analysis of uncoated Avastin® or titanium oxide coated Avastin® compositions (Top): Graph showing the results of size exclusion chromatography analysis of compositions of uncoated Herceptin® or Herceptin® coated with aluminum oxide; (Bottom): quantified monomer and Table showing retention times of aggregates Graph showing the results of ion exchange chromatography analysis of uncoated Herceptin® or aluminum oxide coated Herceptin® compositions Graph showing the results of an SPR binding assay of Avastin® isolated from a composition of uncoated Avastin® or titanium oxide coated Avastin® Table showing KD values quantified from the results shown in Figure 43 Graph showing the results of an SPR binding assay of Herceptin® isolated from a composition of uncoated Herceptin® or Herceptin® coated with aluminum oxide. Table showing KD values quantified from the results shown in Figure 45 Table showing the percentage of Avastin® secondary structure over time as measured by FTIR from uncoated Avastin® or titanium oxide coated Avastin® compositions. Table showing the λmax of Avastin® over time, measured by FTIR from uncoated Avastin® or titanium oxide coated Avastin® compositions. Graph showing the results of Avastin® intrinsic and extrinsic fluorescence over time for compositions of uncoated Avastin® or titanium oxide coated Avastin® Graph showing the percentage of Avastin® aggregates as measured by SEC over time for compositions of uncoated Avastin® or titanium oxide coated Avastin® Table showing the percentage of aggregates quantified from the results presented in Figure 50 Table showing the percentage of secondary structure of Herceptin® over time as determined by FTIR from compositions of uncoated Herceptin® or Herceptin® coated with aluminum oxide. Table showing the λmax of Herceptin® over time as measured by FTIR from compositions of uncoated Herceptin® or Herceptin® coated with aluminum oxide. Graph showing the results of intrinsic and extrinsic fluorescence of Herceptin® over time for compositions of uncoated Herceptin® or Herceptin® coated with aluminum oxide Graph showing the percentage of aggregates of Herceptin® as measured by SEC over time for compositions of uncoated Herceptin® or aluminum oxide coated Herceptin® Table showing the percentage of aggregates quantified from the results presented in Figure 55

The present disclosure provides methods of preparing pharmaceutical compositions that include drugs encapsulated by one or more layers of metal oxides. Such pharmaceutical compositions contain a high fraction of drug that has improved flowability, solubility, stability over time, and is functional before or after administration of the pharmaceutical composition to a subject in need thereof. include. Generally, the provided methods of preparing pharmaceutical compositions can safely, reliably, and predictably produce pharmaceutical compositions having the aforementioned properties. As a result, the provided pharmaceutical compositions and methods of preparing metal oxide encapsulated drugs enhance therapeutic value, increase commercial value, and reduce manufacturing costs per therapeutic dose.

The manufacture of an advantageous pharmaceutical composition consists of sequentially applying a vapor or gaseous metal precursor and a vapor or gaseous oxidizing agent (and using an inert gas after each application of said metal or oxidizing agent). This was made possible by the discovery that by carrying out one or more pump-purge cycles, the entire process could be carried out at lower temperatures, for example not exceeding 35°C. Known methods of coating drugs with metal oxides using vapor or gaseous precursors, when carried out at temperatures below 50°C, reduce the oxidizing agent ( (e.g., water) does not produce a pharmaceutical composition with improved properties. Elevated and sustained levels of oxidant in the reactor can adversely affect the reaction of metal precursors and oxidant with the particle surfaces and with each other (and adsorption). In addition, elevated oxidant levels within the reactor may result from unreacted metal precursors, gaseous by-products from the reaction of the metal precursors with exposed hydroxyl groups on the surface of the substrate or particles, and/or This can impede the ability to remove unreacted oxidants that are not incorporated into the metal oxide layer around the drug, leading to the formation of contaminating metal oxide particles and/or forming around the drug. This can lead to reduced predictability regarding the number and uniformity of metal oxide layers. Without being bound by any particular theory, the pump-purge cycle step is kinetically, rather than thermodynamically, kinetically driven to knock off the oxidant molecules trapped thereon on the particle surfaces and on the interior surfaces of the reactor. It may mediate the effect of As a result, the problematic moisture content within the reactor is less than that expected based on thermodynamic principles well known in the art, as determined by the pressure, temperature, and number of molecules within the reactor. reduced to

Provided herein are methods that utilize mechanical systems and chemical engineering processes. The present disclosure also provides exemplary components and operating conditions for the systems and processes, as well as exemplary drug substrates, vapor and gaseous metal precursors, and vapor and gaseous oxidants.

Drugs The term "drug" in its broadest sense includes small molecules, viral particles, polypeptides, polynucleotides, polypeptides, compositions containing polypeptides and lipids, and compositions containing polynucleotides and lipids. Drugs include analgesics, anesthetics, anti-inflammatory agents, anthelmintic agents, antiarrhythmic agents, anti-asthmatic agents, antibiotics, anti-cancer agents, anti-coagulants, anti-depressants, anti-diabetic agents, anti-epileptic agents, and anti-histamines. Medicines, antitussives, antihypertensives, antimuscarinics, antimycobacteria, antitumor drugs, antioxidants, antipyretics, immunosuppressants, immunostimulants, antithyroid drugs, antivirals, anxiolytic sedatives, hypnotics , neuroleptics, astringents, bacteriostatics, beta-adrenergic receptor blockers, blood products, blood substitutes, bronchodilators, buffering agents, cardiac inotropes, chemotherapeutic agents, contrast media, corticosteroids, cough suppressants Medicines, expectorants, mucolytics, diuretics, dopamine agonists, antiparkinsonian drugs, free radical scavengers, growth factors, hemostatic agents, immunological agents, lipid regulators, muscle relaxants, proteins, peptides, polypeptides, Parasympathomimetic agents, parathyroid calcitonin, bisphosphonates, prostaglandins, radiopharmaceuticals, hormones, sex hormones, antiallergic agents, appetite stimulants, appetite suppressants, steroids, sympathomimetic agents, thyroid agents, vaccines, vasodilators , and xanthine.

Exemplary types of small molecule drugs include, but are not limited to, acetaminophen, clarithromycin, azithromycin, ibuprofen, fluticasone propionate, salmeterol, pazopanib HCl, palbociclib, and amoxicillin-clavulanate potassium. . Exemplary types of polypeptide drugs include proteins (e.g., antibodies), peptide fragments (e.g., antibody fragments), alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamicin, ipilimumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab , rituximab, or trastuzumab. Exemplary types of polynucleotide drugs include messenger mRNA (mRNA), hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, triple helix formation-inducing RNA, aptamers, and one or more of DNA, RNA, including, but not limited to, vectors. Exemplary types of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, and polyketides, and prenol lipids.

In this disclosure, the drug loaded into the reactor can be in powder form. Exemplary methods of preparing drugs in powder form include, but are not limited to, processes utilizing lyophilization, freeze-drying, precipitation, and dry compaction.

Metal Oxide Materials The term "metal oxide materials" in its broadest sense includes all materials formed from the reaction of elements considered to be metals with oxygen-based oxidizing agents. Exemplary metal oxide materials include, but are not limited to, aluminum oxide, titanium oxide, iron oxide, gallium oxide, magnesium oxide, zinc oxide, niobium oxide, hafnium oxide, tantalum oxide, lanthanum oxide, and zirconium dioxide. Not limited. Exemplary oxidizing agents include, but are not limited to, water, ozone, and inorganic peroxides.

Atomic layer deposition (ALD)
Atomic layer deposition is a thin film deposition technique that allows the deposition of films with controlled thickness and uniformity to the level of atomic or molecular monolayers by the sequential addition of self-limiting monolayers of elements or compounds. be. Self-limiting means that only a single atomic layer is formed at a time, and subsequent process steps are required to regenerate the surface and allow further deposition.

Chemical vapor deposition (CVD)
Chemical vapor deposition is a thin film deposition technique in which elements or compounds are deposited onto a surface by chemical reaction in the gas phase or on the surface. It differs from atomic layer deposition in that the deposition is not self-controlled, ie, the film continues to grow as long as the chemicals are supplied. It differs from physical vapor deposition in that the chemical reaction results in a deposited film that is chemically different from the precursor species.

Reactor System The term "reactor system" in its broadest sense includes any system that can be used to perform ALD or mixed ALD/CVD or CVD. An exemplary reactor system is shown in FIG. 1 and described further below.

FIG. 1 shows a reactor system 10 for carrying out the coating of particles, for example heat sensitive particles, in thin film coatings. Reactor system 10 can perform coatings using ALD and/or CVD coating conditions. The relative contribution of ALD and CVD processes to thin film coatings can be controlled by appropriate selection of process conditions. In particular, the reactor system 10 allows primarily ALD processes, eg, substantially entirely ALD processes, to be performed at low processing temperatures, eg, below 50<0>C, such as below 35<0>C. For example, reactor system 10 may primarily form a thin film metal oxide on the particles by ALD at a temperature of 22-35°C, such as 25-35°C, 25-30°C, or 30-35°C. Can be done. Generally, the particles remain or can be maintained at such temperatures. This can be accomplished by holding or maintaining the reactant gases and/or the interior surfaces of the reactor chambers (eg, chamber 20 and drum 40, discussed below) at such temperatures.

By performing the ALD reaction at low temperature conditions, a coating can be formed on the particles without degrading the biological components, such as those of vaccines or biopharmaceuticals. For example, a biological component in amorphous form can be coated without destroying the biological component or converting the biological component to a crystalline form.

Reactor system 10 includes a fixed vacuum chamber 20 coupled to a vacuum pump 24 by a vacuum tube 22. Vacuum pump 24 may be an industrial vacuum pump sufficient to establish a pressure of less than 1 Torr, such as 1 to 100 mTorr, such as 50 mTorr. Vacuum pump 24 allows chamber 20 to be maintained at a desired pressure and allows for removal of reaction by-products and unreacted process gases.

In operation, reactor 10 performs an ALD thin film coating process by introducing gaseous precursors of the coating into chamber 20. Gaseous precursors are alternately spiked into the reactor. This allows the ALD process to be a solvent-free process. The half-reactions of the ALD process are self-limiting and can provide angstrom-level control of deposition. Additionally, ALD reactions can be performed at low temperature conditions, such as below 50°C, such as below 35°C.

Chamber 20 is also coupled to a chemical delivery system 30. Chemical delivery system 20 includes three or more gas sources 32a, 32b, 32c coupled to vacuum chamber 20 by respective delivery tubes 34a, 34b, 34c and controllable valves 36a, 36b, 36c. Chemical delivery system 30 can include a combination of restrictors, gas flow controllers, pressure transducers, and ultrasonic flow meters to provide controllable flow rates of various gases into chamber 20. Chemical delivery system 30 also includes one or more temperature control components, such as, for example, heat exchangers, resistance heaters, heat lamps, etc., to heat or cool the various gases before they enter chamber 20. be able to. Although FIG. 1 shows separate gas lines running parallel to the chamber for each gas source, two or more gas lines, e.g. Can be combined with a valve. Additionally, although three gas sources are shown in FIG. 1, four gas sources may be used to allow in-situ formation of a stacked structure with alternating layers of two different metal oxides.

Two gas sources supply chamber 20 with two chemically different gaseous reactants for the coating process. Suitable reactants include any or combinations of the following: monomer vapors, metal organics, metal halides, oxidizing agents such as ozone or water vapor, and polymeric or nanoparticle aerosols (dry or wet). For example, the first gas source 32a may supply gaseous trimethylaluminum (TMA) or titanium tetrachloride ( _TiCl4 ), whereas the second gas source 32b may supply water vapor. can.

One of the gas sources can supply purge gas. In particular, the third gas source can provide a gas that is chemically inert to the reactants, coating, and particles being treated. For example, the purge gas can be N2 or a noble gas such as argon.

A rotatable coating drum 40 is held within chamber 20 . The drum 40 may be connected to a motor 44 by a drive shaft 42 extending through a sealed port in the side wall of the chamber 20. Motor 44 can rotate the drum at a speed of 1 to 100 rpm. Alternatively, the drum can be connected directly to a vacuum source via a rotating union.

The particles to be coated are shown as a particle bed 50 and are disposed within the interior volume 46 of the drum 40. The drum 40 and chamber 20 can include sealable ports (not shown) that allow particles to be placed in and removed from the drum 40.

The body of drum 40 is provided by one or more of porous material, solid metal, and perforated metal. The pores through the cylindrical side wall of drum 40 may have dimensions of 10 μm.

In operation, as drum 40 rotates, one of the gases flows from chemical delivery system 30 into chamber 20 . The combination of pores (1-100 μm), pores (0.1-10 mm), or large openings in the coating drum allow rapid delivery of precursor chemicals and pumping out of by-products or unreacted nuclides. It serves to trap particles within the coating drum while allowing The pores in the drum 40 allow gas to flow between the exterior of the drum 40 , ie, the reactor chamber 20 and the interior of the drum 40 . In addition, the rotation of drum 40 agitates the particles to keep them separated and ensures that a large surface area of the particles remains exposed. This allows for fast and uniform interaction of the particle surface with the process gas.

In some implementations, one or more temperature control components are incorporated into drum 40 to enable control of the temperature of drum 40. For example, resistive heaters, thermoelectric coolers, or other components may be present in or on the sidewalls of drum 40.

Reactor system 10 also includes a controller 60 coupled to various controllable components, such as, for example, vacuum pump 24, gas distribution system 30, motor 44, temperature control system, etc., to control the operation of reactor system 10. include. Controller 60 may also be coupled to various sensors, such as pressure sensors, flow meters, etc., to provide closed-loop control of the pressure of the gas within chamber 20.

Generally, controller 60 can operate reactor system 10 according to a "recipe." The recipe specifies the operating value of each controllable element as a function of time. For example, the recipe can specify the amount of time the vacuum pump 24 operates, the time and flow rate of each gas source 32a, 32b, 32c, the rotational speed of the motor 44, and the like. Controller 60 can receive the recipe as computer readable data (eg, stored on a non-transitory computer readable medium).

Controller 60 and other computing devices that are part of the systems described herein may be implemented in digital electronic circuitry or computer software, firmware, or hardware. For example, the controller may include a processor executing a computer program stored in a computer program product, such as, for example, a non-transitory machine-readable storage medium. Such a computer program (also known as a program, software, software application, or code) may be written in any form of programming language, including compiled or translated languages, and may also be written as a stand-alone program. , or as modules, components, subroutines, or other units suitable for use in a computing environment. In some implementations, controller 60 is a general purpose programmable computer. In some implementations, the controller may be implemented using dedicated logic circuits, such as, for example, FPGAs (Field Programmable Gate Arrays) or ASICs (Application Specific Integrated Circuits).

Operation Initially, particles are loaded into the drum 40 of the reactor system 10. The particles can have a solid core containing a drug, such as one of the drugs mentioned above. Once the access port is sealed, controller 60 operates reactor system 10 according to the recipe to form a thin metal oxide layer on the particles.

In particular, two reactant gases are alternately supplied to the chamber 20, and following each step of supplying the reactant gases, an inert gas is supplied to the chamber 20 to drive out the reactant gases and by-products used in the previous step. A purge cycle is performed. Furthermore, the one or more gases (e.g., reactive gases and/or inert gases) can be supplied in pulses, where chamber 20 is filled with gas to a specified pressure and a delay time is reached. Allow time to elapse and the chamber is evacuated by vacuum pump 24 before the next pulse begins.

In particular, controller 60 may operate reactor system 10 as follows.

In the first reactant half cycle, motor 44 rotates drum 40 to agitate particles 50 while
i) Gas distribution system 30 is operated to flow a first reactant gas, eg, TMA, from gas source 32a into chamber 20 until a first specified pressure is achieved. The specific pressure may be 0.1 Torr for half the saturation pressure of the reactant gas.
ii) The flow of the first reactant is stopped and a specified delay time is allowed to elapse, for example as measured by a timer in the controller. This allows the first reactant to flow through the bed of particles within drum 40 and react with the surface of particles 50 within drum 40 .
iii) Vacuum pump 50 evacuates chamber 20 such that the pressure is reduced, eg to less than 1 Torr, eg 1-100 mTorr, eg 50 mTorr.

These steps (i) to (iii) can be repeated a number of times set by the recipe, for example 2 to 10 times, for example 6 times.

Next, during a first purge cycle, motor 44 rotates the drum to agitate particles 50 while:
iv) The gas distribution system 30 is operated to flow an inert gas, eg, N2, into the chamber 20 from the gas source 32c until a second specified pressure is achieved. The second specified pressure can be between 1 and 100 Torr.
v) The flow of inert gas is stopped and a specified delay time is allowed to elapse, for example as measured by a timer in the controller. This allows inert gas to flow through the pores of drum 40 and diffuse through particles 50 to displace reactant gas and any vapor byproducts.
vi) Vacuum pump 50 evacuates chamber 20 such that the pressure is reduced, eg to less than 1 Torr, eg 1-500 mTorr, eg 50 mTorr.

These steps (iv) to (vi) can be repeated a number of times set by the recipe, for example 6 to 20 times, for example 16 times.

In the second reactant half cycle, motor 44 rotates drum 40 to agitate particles 50 while
vii) The gas distribution system 30 is operated to flow a second reactant gas, e.g. H2O, into the chamber 20 from the gas source 32a until a third specified pressure is achieved. The third pressure may be 0.1 Torr for half the saturation pressure of the reactant gas.
viii) The flow of the second reactant is stopped and a specified delay time is allowed to elapse, for example as measured by a timer in the controller. This allows the second reactant to flow through the pores in the drum 40 and react with the surface of the particles 50 within the drum 40.
ix) The vacuum pump 50 evacuates the chamber 20 such that the pressure is reduced, eg to less than 1 Torr, eg 1 to 500 mTorr, eg 50 mTorr.

These steps (vii) to (ix) can be repeated a number of times set by the recipe, for example 2 to 10 times, for example 6 times.

A second purge cycle is then performed. This second purge cycle may be the same as the first purge cycle or may have a different number of repetitions of steps (iv)-(vi) and/or a different delay time and/or a different pressure. can.

The cycles of first reactant half-cycle, first purge cycle, second reactant half-cycle, and second purge cycle can be repeated a number of times set by the recipe, e.g., from 1 to 10 times. .

As mentioned above, the coating process can be carried out at low processing temperatures, such as less than 50°C, such as 35°C or less. In particular, the particles may remain or be maintained at such temperature during all steps (i) to (ix) above. Generally, the temperature inside the reactor chamber does not exceed 35° C. during steps (i) to (ix). This can be accomplished by injecting the first reactant gas, the second reactant gas, and the inert gas into the chamber at such temperatures during each cycle. In addition, the physical components of the chamber can be kept at or maintained at such temperatures, if desired, using, for example, a cooling system, such as a thermoelectric cooler.

Process for Preparing Pharmaceutical Compositions Comprising a Drug Encapsulated by One or More Layers of Metal Oxide Two Exemplary Examples of Pharmaceutical Compositions Comprising a Drug-Containing Core Surrounded by One or More Metal Oxide Materials method is provided. A first exemplary method includes the following series of steps: (a) loading drug-containing particles into a reactor; (b) applying a vapor or gaseous metal precursor to a substrate within the reactor. (c) performing one or more pump purge cycles of the reactor using an inert gas; (d) applying a vapor or gaseous oxidant to the substrate within the reactor; and (e) Performing one or more pump purge cycles of the reactor using an inert gas. While carrying out the method, the temperature of the particles does not exceed 35°C.

In some implementations of the first exemplary method, the series of steps (b)-(e) includes increasing the total thickness of the one or more metal oxide materials surrounding the solid core of the coated particle. Optionally repeated one or more times to increment. In some embodiments, the reactor pressure can stabilize following step (a), step (b), and/or step (d). In some embodiments, the contents of the reactor are agitated before and/or during step (b), step (c), and/or step (e). In some embodiments, a subset of the vapor or gaseous contents is pumped out before step (c) and/or step (e).

A second exemplary method includes (a) loading drug-containing particles into a reactor, (b) reducing reactor pressure to less than 1 Torr, and (c) until the contents of the reactor have a desired water content. agitating the contents of the reactor; (d) pressurizing the reactor to at least 10 Torr by adding steam or gaseous metal precursor; (e) stabilizing the reactor pressure; (f) the contents of the reactor. (g) pumping out a subset of the vapor or gaseous contents and metal precursors and metal precursor by-products that react with exposed hydroxyl residues on the substrate or on the particle surface; (h) performing a series of pump purge cycles of the reactor using an inert gas; i) pressurizing the reactor to at least 10 Torr by adding steam or gaseous oxidizing agent; (j) stabilizing the reactor pressure; (k) stirring the reactor contents; (l) steam or gas; pumping out a subset of the contents of the particles, including metal precursors that react with exposed hydroxyl residues on the substrate or on the particle surface, by-products of the metal precursors, and unreacted oxidizing agents. (m) performing a series of pump purge cycles of the reactor using an inert gas; including (e.g. consisting of). While carrying out the method, the temperature of the particles does not exceed 35°C.

In some embodiments of the second exemplary method, the series of steps (b)-(m) includes increasing the total thickness of the one or more metal oxide materials surrounding the solid core of the coated particle. Optionally repeated one or more times to increment.

Pharmaceutically Acceptable Additives, Diluents, and Carriers Pharmaceutically acceptable excipients include, but are not limited to:
(1) Surfactants and polymers, including: polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), sodium lauryl sulfate, polyvinyl alcohol, crospovidone, polyvinylpyrrolidone-polyvinyl acrylate copolymer, cellulose derivatives, hydroxypropyl methylcellulose , hydroxypropylcellulose, carboxymethylethylcellulose, hydroxypropylmethylcellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols, carbomers and their polymers, emulsifiers, sugar gums, starches, organic acids and their salts, vinylpyrrolidone and Vinyl acetate;
(2) Binders, such as cellulose, cross-linked polyvinylpyrrolidone, microcrystalline cellulose, etc.;
(3) fillers, such as lactose monohydrate, anhydrous lactose, microcrystalline cellulose, and various starches;
(4) Lubricants, such as agents that affect the fluidity of the powder to be compacted, including colloidal silicon dioxide, talc, stearic acid, magnesium stearate, calcium stearate, silica gel;
(5) Sweeteners, any natural or artificial sweeteners such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K;
(6) Flavoring agent;
(7) Preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, and phenols such as phenol. compounds, or quaternary compounds such as benzalkonium chloride;
(8) Buffer;
(9) diluents such as pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars, and/or mixtures of any of the foregoing;
(10) Wetting agents, such as corn starch, potato starch, corn starch, modified starch, and mixtures thereof;
(11) disintegrants; for example, croscarmellose sodium, crospovidone, sodium starch glycolate; and (12) blowing agents, for example, organic acids (for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, and anhydrides and acid salts), or carbonates (e.g., sodium carbonate, potassium carbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate), or bicarbonates ( effervescent couples, such as sodium bicarbonate or potassium bicarbonate.

The following materials and methods were used in the examples described herein.

Example 1: Preparation of particles containing a drug encapsulated with a uniform and thin aluminum oxide coating layer with nanometer precision at a process temperature below 35 °C In this example, we prepare a drug encapsulated with a metal oxide. One of the disclosed methods is performed and the data presented. In this example, the vapor or gaseous metal precursor is trimethylaluminum (TMA), and after the TMA reacts with the exposed hydroxyl groups on the particles or on the surface of the coated particles, the by-product gaseous methane is is formed and the oxidizing agent is water vapor.

Method Briefly, the method consists of the following series of steps:
(a) loading particles containing a drug into a reactor;
(b) reducing the reactor pressure to less than 1 Torr;
(c) agitating the contents of the reactor until the contents of the reactor reach the desired moisture content by performing a residual gas analysis (RGA) to monitor the level of water vapor within the reactor;
(d) pressurizing the reactor to at least 1 Torr by adding steam or gaseous TMA;
(e) stabilizing the reactor pressure;
(f) agitating the contents of the reactor;
(g) RGA for pumping out a subset of the vapor or gaseous contents, including gaseous methane and unreacted TMA, and for monitoring the levels of gaseous methane and unreacted TMA in the reactor; determining when to stop pumping by performing;
(h) performing a series of pump purge cycles on the reactor using nitrogen gas;
(i) pressurizing the reactor to at least 1 Torr by adding water vapor;
(j) stabilizing the reactor pressure;
(k) agitating the contents of the reactor;
(l) Pumping a subset of the steam or gaseous contents, including water vapor, and knowing when to stop pumping by running an RGA to monitor the level of water vapor in the reactor. to decide;
(m) Performing a series of pump purge cycles on the reactor using nitrogen gas.

While carrying out the method, the internal temperature of the reactor did not exceed 35°C. Additionally, steps (b) to (m) were repeated more than once to increase the overall thickness of aluminum oxide surrounding the solid core. FIG. 2 includes representative process conditions for carrying out this method.

Results Figure 3 shows representative residual gas analysis traces measured during steps (d), (h), (i), and (m) for one cycle of the method. This method reproducibly exhibits metal oxide coating growth rates of 2 to 4 angstroms per cycle. In contrast, another method that limited growth to ALD showed average growth of only 1 angstrom per cycle. Without being bound by any particular theory, given the growth rates observed with this method, growth may be mediated by a combination of ALD and CVD.

Example 2: Determining whether encapsulation of small molecules with metal oxide coatings changes structure or solubility profile To assess whether encapsulation of small molecules with metal oxide coatings changes structure or solubility profile small molecule clarithromycin, clarithromycin carbomer complex, pazopanib HCl. Palbociclib and potassium amoxicillin-clavulanate were encapsulated with a metal oxide coating, and the resulting particles were subjected to chemical analysis to determine whether the metal oxide coating altered the structure or dissolution profile. Small molecules in powder form were encapsulated by the method provided in this disclosure with the following modifications as shown in the table below.

Method High Performance Liquid Chromatography Samples at a concentration of 1 mg/mL were prepared by dissolving the analyte in a 50:50 volume:vol mixture of acetonitrile and water and filtered through a 0.45 μm filter. The exact conditions of analysis, including mobile phase, column, and oven temperature will vary depending on the analyte being considered. A typical example of analysis is 0.05 M pH 4 phosphate buffer mixed with acetonitrile (90:10 v/v), Agilent Pursuit XRs 3C-18 3 μm column, 37°C oven temperature, flow rate 0.9 mL/min, Use a mobile phase consisting of an injection volume of 35 μL, a run time of 5 minutes, and a UV detector at 214 nm.

Matrix-assisted laser desorption ionization coupled to mass spectrometry (MALDI-MS)
Samples were prepared by dissolving in a water-acetonitrile mixture at a ratio of 18:82 v/v. The sample was then mixed with a cyano-4-hydroxycinnamic acid matrix and loaded onto a MALDI chip. MS data acquisition was performed in reflectron positive mode.

Results The results are shown in Figures 4-14. Compared to uncoated controls, small molecule clarithromycin and clarithromycin carbomer complexes coated with either titanium oxide or aluminum oxide showed little or no change in structure as detected by HPLC analysis. did not show. Small molecule pazopanib HCl. coated with either titanium oxide or aluminum oxide compared to uncoated controls. and palbociclib showed little or no change in structure as detected by MALDI-MS analysis. Small molecule clarithromycin, clarithromycin carbomer complex, and palbociclib coated with either titanium oxide or aluminum oxide, and pazopanib HCl coated with aluminum oxide compared to uncoated controls. It showed little or no change in dissolution profile. In contrast, small molecule pazopanib HCl coated with titanium oxide and potassium amoxicillin clavulanate coated with either titanium oxide or aluminum oxide exhibited altered dissolution profiles compared to uncoated controls. Indicated. Pazopanib HCl coated with titanium oxide exhibits an altered dissolution profile (similar initial release and subsequent delayed release) compared to the uncoated control. Potassium amoxicillin clavulanate coated with titanium oxide exhibits an altered dissolution profile (slow initial release followed by rapid release without saturation for 30 minutes) compared to the uncoated control. Finally, amoxicillin clavulanate potassium coated with aluminum oxide exhibits an altered dissolution profile (slow initial release followed by rapid release) compared to the uncoated control.

Conclusion:
This example shows that encapsulation of five small molecules with either titanium oxide or aluminum oxide does not significantly reduce the small molecule structure, but does change the dissolution profile depending on the small molecule and/or metal oxide coating. have been demonstrated to cause little or no change or significant change in Without being bound by any particular theory, Applicants believe that, overall, 1) the effects on dissolution profiles can vary widely for the same coating material (based on process conditions, API, and dissolution conditions); It is surprising to note that, and 2) coatings made of the same base material may have essentially no effect on the dissolution profile. This means a versatile process that can use the same base material to create melt profiles tailored to specific applications. Applicants have determined that, as described herein, those skilled in the art will test various methods or parameters to ensure that the structure is not significantly reduced and that the dissolution compared to uncoated small molecules We conclude that it is possible to produce small molecules coated with selected metal coatings that exhibit or do not exhibit profile changes.

Example 3: Determining whether encapsulation of small molecules with metal oxide coatings retards crystallization of amorphous indomethacin exposed to moisture. Encapsulation of small molecules with metal oxide coatings alters structure or dissolution profile. To assess whether the small molecule indomethacin was encapsulated with a metal oxide coating and the resulting particles were subjected to chemical analysis, we determined whether the metal oxide coating changed the crystallinity of amorphous indomethacin. It was determined. Small molecules in powder form were encapsulated by the method provided in this disclosure with the following modifications as shown in the table below.

Methods Evaluation of crystallinity of amorphous indomethacin Amorphous indomethacin was prepared by freeze-drying a standard indomethacin sample, and the crystal content before and after exposure to moisture was determined by differential scanning calorimetry (DSC). The area under the crystallization peak was used to determine the specific heat of crystallization of the amorphous material. The percent crystallinity of a partially crystalline material was determined by dividing the heat of crystallization by the heat of crystallization of a fully amorphous material, subtracting that value from 1, and multiplying by 100.

Results Compared to the uncoated control, small molecule indomethacin coated with either titanium oxide or aluminum oxide was amorphous in the as-treated state and after exposure to 90% relative humidity (RH). showed a decrease in the conversion from the crystalline state to the crystalline state (FIG. 15).

Conclusion:
The present example demonstrates that the provided methods specifically demonstrate that the drug in the coated particles is removed from the amorphous state in the as-processed state and after exposure to stress, such as 90% RH. We provide guidance that small molecules coated with more stable metal oxide materials can be produced that exhibit reduced conversion to the crystalline state.

Example 4: Determining whether the provided methods enable uniform, conformal, thin metal oxide coatings on small molecules. The provided methods enable uniform, conformal, thin metal oxide coatings on small molecules. To assess whether acetaminophen could be coated with a metal oxide material by the method of the present disclosure and analyzed by atomic layer microscopy and XPS analysis. Small molecules in powder form were encapsulated with the methods provided in this disclosure.

Methods Transmission Electron Microscopy Samples for TEM were prepared using in situ FIB lift-out technology on a FEI Strata 400 Dual Beam FIB/SEM. Before milling, the samples were capped with protective carbon and e-Pt/I-Pt. The thickness of the TEM lamella was approximately 100 nm. The samples were imaged on a FEI Tecnai TF-20 FEG/TEM operating at 200 kV in bright field (BF) TEM mode and high resolution ((HR) TEM mode. Using energy dispersive spectroscopy (EDS), Qualitative elemental maps of the images were obtained.

XPS Analysis X-ray photoelectron spectroscopy (XPS) was performed on the samples to obtain details of the surface chemistry before and after coating. Powder samples were attached to adhesive substrates and loaded into the instrument. Soft X-rays (1486 eV) were used to excite the sample, the X-ray penetration depth was 5 nm, and the spot size was 200 μm.

Results Direct TEM imaging of cross-sections prepared by focused ion beam (FIB) milling of coated acetaminophen particles reveals uniform and conformal formation of drug particles with aluminum oxide at the nanometer scale, regardless of their position on the particle. (Figure 16). Energy dispersive spectroscopy (EDS) qualitatively demonstrated that the coating consisted essentially of aluminum and oxygen (Figure 17).

Conclusion:
This example provides guidance that the provided method enables uniform and conformal thin metal oxide coatings on small molecules with nanometer-level precision.

Example 5. Determining whether encapsulation of a lyophilized monoclonal antibody (mAb) with a metal oxide coating changes the structure, stability, or ability of the mAb to bind to a target polypeptide. Encapsulation of a lyophilized mAb with a metal oxide coating The mAbs trastuzumab (Herceptin®) and bevacizumab (Avastin®) were encapsulated with a metal oxide coating and the resulting particles Biochemical and chemical analyzes were submitted to determine whether the metal oxide coating altered the mAb's structure, stability, or ability to bind to the target polypeptide. Two mAbs were encapsulated by the method provided in this disclosure with the following modifications: (1) for Herceptin®, mixing the coated particles with a pharmaceutically acceptable diluent or carrier; In the previous series of 99 cycles, the vapor or gaseous metal precursor was aluminum oxide (Al ₂ O ₃ ); (2) for Avastin®, the coated particles were The vapor or gaseous metal precursor was titanium oxide (TiO ₂ ) for 49 successive cycles before mixing with a diluent or carrier.

Method Liquid chromatography combined with mass spectrometry Agilent using mobile phase A (0.1% (v/v) FA) and 10% mobile phase B (0.1% (v/v) FA in acetonitrile) An AdvanceBio RP mab C4 (Agilent Technologies) column was analyzed using an Agilent 1260 Infinity Bio-inert Quaternary LC system connected to a 6230 electrospray ionization time-of-flight mass spectrometer (ESITOF-MS) instrument. Reversed phase chromatography (RPC) ) was carried out. Samples were buffer exchanged via a 10 kDa MWCO Centricon (Pall Corporation), loaded onto the column, and separated using a linear gradient of 10%-65% B at a flow rate of 0.5 ml/min. MS spectra were calibrated in positive ion mode and TIC was recorded from 1,000 to 7000 m/z. Capillary gas temperature/voltage (Vcap) was set at 350° C./5,500V, respectively, and fragmentor voltage (Vfrag) was 400V. MS spectra were deconvolved using the maximum entropy (MaxEnt) algorithm as part of Agilent MassHunter Qualitative Analysis and BioConfirm software.

Peptide mapping Connected to Agilent 6230 ESI-TOF-MS instrument using mobile phase A (0.1% (v/v) TFA) and mobile phase B (0.1% (v/v) TFA in acetonitrile) Reversed phase chromatography (RPC) was performed on an AdvanceBio Peptide Mapping C18 (Agilent Technologies) column operated at 55°C using an Agilent 1260 Infinity Bio-inert Quaternary LC system. Digested samples were injected onto the column and separated using a linear gradient from 5% to 65% B at a flow rate of 0.3 ml/min. MS spectra were calibrated in positive ion mode and TIC was recorded from 100 to 3200 m/z. Capillary gas temperature/Vcap were set to 300°C/4500V, respectively, and Vfrag was 300V. MS spectra were analyzed using Agilent MassHunter qualitative analysis and the Protein Molecular Feature Extraction (MFE) algorithm in BioConfirm software to obtain a list of potential peptides, which were matched against in-silico digested mAb peptides. , sequence coverage was obtained.

Fourier transform infrared spectroscopy (FTIR)
FTIR spectra were recorded at room temperature using the attenuated total reflectance (ATR) method. FTIR absorbance spectra of 0.5 mg/ml mAb were collected in the range 500-4000 cm ⁻¹ . The second derivative spectrum was obtained by applying an 11-point Savitsky-Golay smoothing of the original spectrum. The second-order differential spectrum in the 700 cm ^-1 range with 1600 Savitzky-Golay smoothing was deconvoluted by a curve fitting method using the Levenberg-Marquardt algorithm, and α-helices (1660-1654 cm ^-1 ), β-sheets ( The peaks corresponding to 1637 to 1614 cm ^-1 ), turns (1678 to 1670 cm ^-1 ), random coils (1648 to 1638 cm ^-1 ), and β-antiparallel (1691 to 1680 cm ^-1 ) were adjusted, and a Gaussian function was used to The area was measured. The areas of all component bands assigned to a given conformation were then summed and divided by the total area.

Circular dichroism (CD)
Deep UV CD spectra were recorded in the range 200-250 nm at 25° C. with a spectral bandwidth of 5 nm using a quartz cell with a path length of 0.1 cm at a scanning speed of 50 nm/min. The sample concentration was maintained at 0.2 mg/ml and three spectra were scanned, averaged and finally plotted after buffer baseline subtraction. The mean residue ellipticity (MRE, deg cm2/dmole) was calculated.

Fluorescence spectroscopy Intrinsic fluorescence was measured by exciting protein solutions (0.5 mg/ml) at 295 nm (tryptophan excitation only). Emission spectra were recorded in the range 300-450 nm. Record the external fluorescence intensity (0.5 mg/ml) of the sample using a fluorescence spectrophotometer using ANS (8-anilinonaphthalene-1-sulfonic acid) dye with excitation at 380 nm and emission between 400 and 600 nm. did. All measurements were performed in triplicate. Each spectrum represents the average of three scans.

Size exclusion chromatography (SEC-HPLC)
Size exclusion chromatography was performed on a Superdex 200 column using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) with a buffer of 300 mM NaCl and 0.05% NaN3 at pH 6.8. Detection was performed by monitoring UV absorbance at 280 nm. Peak integrals and peak areas were determined using Chromeleon software.

Cation exchange chromatography (CEX)
Ion exchange chromatography was performed using a Dionex Ultimate 3000 RSLC system (Thermo Scientific) with an Agilent Bio MAb NP5 column and a buffer of 300 mM NaCl and 0.05% NaN3 at pH 6.8. Detection was performed by monitoring UV absorbance at 280 nm.

Surface Plasmon Resonance (SPR) FcRn Binding Kinetic Assay Binding kinetic interactions of various mAb samples to human FcRn receptors were determined using HBS-EP buffer (GE Healthcare Life Sciences) in Biacore X100™ (GE Healthcare). Measurements were made using surface plasmon resonance. Recombinant human FcRn antibodies were immobilized and samples were injected at a series of concentrations. Rate constants were calculated from sensorgrams using a 1:1 fit model using BIA Evaluation 2.0.1 software.

Stability analysis To determine the structural integrity and aggregation of the metal oxide coated mAb over 10 days at 80 °C, the higher order structure of the mAb was measured by Fourier transform infrared spectroscopy (FTIR); The tertiary structure of the mAb was determined by intrinsic and extrinsic fluorescence analysis, and the size variant profile was determined by size exclusion chromatography (SEC). Samples were stored in a dry bath at 80° C. and sampled at set times for testing. FTIR spectra were recorded at room temperature using the attenuated total reflectance (ATR) method. FTIR absorbance spectra of 0.5 mg/ml mAb were collected in the range 500-4000 cm ⁻¹ . The second derivative spectrum was obtained by applying an 11-point Savitsky-Golay smoothing of the original spectrum. The second-order differential spectrum in the range of 1600 to 1700 cm ^-1 was deconvoluted by a curve fitting method using the Levenberg-Marquardt algorithm, and α-helices (1660 to 1654 cm ^-1 ), β-sheets (1637 to 1614 cm ^{- 1} ), peaks corresponding to turns (1678 to 1670 cm ^-1 ), random coils (1648 to 1638 cm ^-1 ), and β-antiparallel (1691 to 1680 cm ^-1 ) were adjusted, and the area was measured using a Gaussian function. . The areas of all component bands assigned to a given conformation were then summed and divided by the total area. Intrinsic fluorescence was measured by exciting the protein solution (0.5 mg/ml) at 295 nm (tryptophan excitation only). Emission spectra were recorded in the range 300-450 nm. Record the external fluorescence intensity (0.5 mg/ml) of the sample using a fluorescence spectrophotometer using ANS (8-anilinonaphthalene-1-sulfonic acid) dye with excitation at 380 nm and emission between 400 and 600 nm. did. All measurements were performed in triplicate. Each spectrum represents the average of three scans. Size exclusion chromatography was performed as described above.

Results Liquid chromatography combined with mass spectrometry was performed to confirm the mass and sequence identity of the mAb encapsulated in the metal coating. Results for Avastin® are shown in Figures 18-20. The data suggest that the titanium oxide coating has little or no effect on the intact protein mass of Avastin®. The predicted changes for coated Avastin® were consistent with those for uncoated Avastin®, except for one change. Results for Herceptin® are shown in Figures 21-23. The data suggest that the aluminum oxide coating has little or no effect on the intact protein mass of Herceptin® compared to uncoated Avastin®.

Peptide mapping was performed to confirm sequence identity and post-translational modifications of mAbs encapsulated with metal coatings. Results for Avastin® are shown in Figures 24-25. The data suggest that the titanium oxide coating has little or no effect on the sequence identity of Avastin® when compared to in-silico digested Avastin®.

The results for Herceptin® are shown in Figures 26-27. The data suggest that the aluminum oxide coating has little or no effect on the sequence identity of Herceptin® when compared to in-silico digested Herceptin®.

Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) analysis were performed to determine if there were any changes in the secondary structure of the mAb. Fluorescence spectroscopy was performed to determine if there was a change in the tertiary structure of the mAb. Results for Avastin® are shown in Figures 28-33. The data suggest that the titanium oxide coating has little or no effect on the secondary structure of Avastin® when compared to uncoated Avastin®. Results for Herceptin® are shown in Figures 34-38. Data suggest that the aluminum oxide coating has little or no effect on the secondary structure of Herceptin® compared to uncoated Herceptin®, as determined by FTIR. . The data show that aluminum oxide has little or no effect on the tertiary structure of Herceptin® compared to uncoated Herceptin®, as detected by far-UV and near-UV circular dichroism analysis. This suggests that it has no effect.

Size exclusion chromatography (SEC) was performed to determine whether there were changes in the size variants of the mAb samples. Cation exchange chromatography (CEX) was performed to determine if there was a change in the charge variant profile of the mAb samples. Results for Avastin® are shown in Figures 39-40. The data show that the titanium oxide coating has little or no effect on the percentage of monomers and aggregates or the percentage of charge variants of Avastin® compared to uncoated Avastin®. Suggests. Results for Herceptin® are shown in Figures 40-41. The data show that the aluminum oxide coating has little or no effect on the percentage of monomers and aggregates or the percentage of charge variants of Herceptin® compared to uncoated Herceptin®. Suggests.

To determine mAb functionality, binding rates were determined by surface plasmon resonance (SPR). Results for Avastin® are shown in Figures 43-44. Data show that titanium oxide coated Avastin® has 4 times stronger binding (reflected by a reduction in KD) to targeted human FcRn receptors compared to uncoated Avastin®. This suggests that the Results for Herceptin® are shown in Figures 45-46. Data show that Herceptin® coated with aluminum oxide has similar binding (reflected by similar KD values) to targeted human FcRn receptors compared to uncoated Herceptin®. This suggests that

To determine the structural integrity and aggregation of the metal oxide-coated mAb over 10 days at 80 °C, the secondary structure of the mAb was measured by Fourier transform infrared spectroscopy (FTIR), and the tertiary structure of the mAb was determined by Fourier transform infrared spectroscopy (FTIR). was measured by intrinsic and extrinsic fluorescence analysis, and the profile of size variants was determined by size exclusion chromatography (SEC). Results for Avastin® are shown in Figures 47-51. The data show that titanium oxide coated Avastin® compared to uncoated Avastin® at the level of mAb secondary or tertiary structure detected by FTIR and fluorescence analysis, respectively. This suggests little or no change. The data also suggest that Avastin® coated with titanium oxide exhibits a reduced rate of aggregate accumulation, as determined by SEC, compared to uncoated Avastin®. ing. Results for Herceptin® are shown in Figures 52-56. Data show that Herceptin® coated with titanium oxide compared to uncoated Herceptin® at the level of mAb secondary or tertiary structure detected by FTIR and fluorescence analysis, respectively. This suggests little or no change. The data also suggest that Herceptin® coated with titanium oxide exhibits similar rates of aggregate accumulation, as determined by SEC, compared to uncoated Herceptin®. ing.

Conclusion:
This example demonstrates that encapsulation of two different lyophilized mAbs with a metal coating does not result in a significant decrease in the structure, stability, or ability of the mAbs to bind to a target polypeptide (e.g., human FcRn). are doing. Notably, titanium oxide coated Herceptin® has four times stronger binding (KD of (reflected by a reduction). Also of note is that Avastin(R) coated with titanium oxide showed less accumulation of aggregates at 80°C for 10 days as determined by SEC compared to uncoated Avastin(R). It is to show a decrease in the rate. Applicants have determined that those skilled in the art can test the various methods or parameters described herein to determine which mAbs are coated with a selected metal coating that does not significantly reduce mAb structure, stability, or ability to bind to a target polypeptide. We conclude that it is possible to produce lyophilized mAbs.

Claims (17)

1. A method of preparing a pharmaceutical composition comprising a drug-containing core surrounded by one or more metal oxide materials, the method comprising:
(a) loading the reactor with particles containing the drug and having a median particle size between 0.1 μm and 1000 μm based on a volume average;
(b) applying a vapor or gaseous metal precursor to the particles in the reactor;
(c) performing one or more pump purge cycles of the reactor using an inert gas;
(d) applying steam or gaseous water as an oxidizing agent to the particles in the reactor;
(e) performing one or more pump purge cycles of the reactor using an inert gas; and (f) repeating steps (b) to (e) one or more times to surround the core. comprising a series of steps of increasing the overall thickness of the metal oxide material;
The particles remain in the reactor during the repeated steps, each pump purge cycle including injecting inert gas into the reactor chamber until the desired pressure is reached, and after a delay time, the inert gas pressure is 1 torr. pumping inert gas from the reactor until the pressure is below 1 torr; and flowing inert gas into the reactor chamber until the desired pressure is reached; and after a delay period, until the pressure of the inert gas is below 1 torr. repeating the step of pumping the inert gas out of the reactor, such that the temperature of the particles does not exceed 35°C, thereby forming a drug-containing core surrounded by one or more metal oxide materials. producing a pharmaceutical composition comprising;
the one or more metal oxide materials are selected from the group consisting of aluminum oxide, titanium oxide, and zinc oxide;
Method. 2. The method of claim 1, wherein the reactor pressure is stabilized at less than 1 Torr for step (a), at least 1 Torr for step (b), and /or at least 1 Torr for step (d). 2. A method according to claim 1, wherein a subset of the vapor or gaseous contents is pumped out before step (c) and/or step (e). 2. The method of claim 1, wherein the particles include a drug and one or more pharmaceutically acceptable excipients. 2. The method of claim 1, wherein the particles have a median particle size of between 0.1 [mu]m and 1000 [mu]m, based on a volume average. 2. The method of claim 1, wherein the pharmaceutical composition is removed from the reactor and mixed with a pharmaceutically acceptable diluent or carrier. 2. The method of claim 1, wherein the particles consist essentially of drug. 2. The method of claim 1, wherein the drug is a small molecule, a viral particle, a polypeptide, a polynucleotide, a composition comprising a polypeptide and a lipid, or a composition comprising a polynucleotide and a lipid. 2. The method of claim 1, wherein the one or more metal oxide materials are selected from the group consisting of aluminum oxide and titanium oxide. 2. A method according to claim 1, wherein the contents of the reactor are agitated before and/or during step (b), step (c) and/or step (e). A method according to claim 1, wherein the temperature of the particles remains between 22°C and 35°C. The method according to claim 1, wherein the inert gas is nitrogen gas or a rare gas. 13. The method according to claim 12, wherein the inert gas is nitrogen gas. Claim 1, wherein the drug is a small molecule drug selected from the group consisting of acetaminophen, clarithromycin, azithromycin, ibuprofen, fluticasone propionate, salmeterol, pazopanib HCl, palbociclib, and amoxicillin-clavulanate potassium. The method described. 2. The method of claim 1, wherein the drug is a small molecule drug selected from the group consisting of clarithromycin, clarithromycin carbomer complex, pazopanib HCl, palbociclib, and amoxicillin-clavulanate potassium. 2. The method of claim 1, wherein step (c) is repeated 6 to 20 additional times. The method of claim 1, wherein step (e) is repeated from 1 to 10 additional times.

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